Introducing higher harmonic generation microscopy for renal tumour pathology: a perspective on clinical translation

Abstract Higher harmonic generation microscopy (HHGM) is an emerging nonlinear optical imaging modality that combines second harmonic generation (SHG), third harmonic generation (THG), and two-photon excited autofluorescence (2PEF) to generate high-resolution, label-free images of fresh biological tissue. By exploiting intrinsic optical contrast arising from nonlinear light–tissue interactions, HHGM enables rapid visualisation of cellular morphology, extracellular matrix architecture, and the tumour microenvironment without fixation or staining. These unique biophysical properties have facilitated the clinical translation of HHGM for real-time tissue assessment across several oncological applications. Renal tumour pathology represents a promising yet underexplored application of HHGM, particularly because rapid assessment of biopsy adequacy and tumour representativeness remains an important unmet clinical need. In this perspective, we discuss the biophysical principles of HHGM, review the current evidence supporting its application in oncological pathology, and present representative images that illustrate the characteristic multimodal appearance of renal cell carcinoma (RCC) and normal renal parenchyma, highlighting qualitative correspondence with conventional haematoxylin and eosin histology. In addition, HHGM enables visualisation of stromal architecture and the tumour microenvironment in fresh, unprocessed tissue within minutes. Although the ongoing prospective Intelligence Imaging (INTIMA) study is designed to validate the diagnostic accuracy, reproducibility, and clinical utility of HHGM in renal tumour pathology, these findings should be considered preliminary until such validation is completed. If confirmed, together with continued advances in nonlinear optical microscopy, digital pathology, and artificial intelligence (AI), HHGM could become a valuable adjunct to routine histopathological assessment and real-time biopsy evaluation.